The present invention relates generally to hybridomas capable of producing antibodies, even more particularly to antibodies specific for cell surface antigens, and even more particularly to the methods for utilizing the antibodies, the cells they identify, and the antigens with which they interact.
The immune system is crucial for survival, as it initiates the destruction and elimination of both invading organisms (e.g., a virus) and toxic materials produced by such organisms. There are two broad types of immune system responses: (1) xe2x80x9cantibodyxe2x80x9d responses; and (2) xe2x80x9ccell mediatedxe2x80x9d responses. Antibodies circulate in the bloodstream and permeate other body fluids and tissues. Antibodies generally bind with specificity to the antigens which induced the antibody response. This binding works to both inactivate the antigen as well as aid in the destruction of the antigen. The cell mediated response involves the production of specialized cells which react with antigens on the surface of virus-infected host cells. These specialized cells work to destroy the virus-infected host cell before the virus is able to replicate and further infect other cells.
The cells involved in the immune system response are a class of white blood cells referred to as xe2x80x9clymphocytes.xe2x80x9d These lymphocytes are generally classified into two types: xe2x80x9cB cellsxe2x80x9d and xe2x80x9cT cells.xe2x80x9d B cells are generally described as having xe2x80x9creceptorsxe2x80x9d which can xe2x80x9crecognizexe2x80x9d an antigen alone, leading to production of antibodies specific for the recognized antibody. T cells, on the other hand, are known to have receptors which cannot recognize an antigen unless it is associated with cell-membrane proteins referred to as xe2x80x9cmajor histocompatability complexxe2x80x9d (MHC) molecules. There are generally two classes of MHC molecules, referred to as xe2x80x9cclass Ixe2x80x9d and xe2x80x9cclass IIxe2x80x9d. In the cell mediated response, the antigen is degraded by the cell, forming small peptide fragments that form physical complexes with a xe2x80x9cclass Ixe2x80x9d or xe2x80x9cclass IIxe2x80x9d MHC molecule. The peptide-class I or peptide-class II MHC complex is then exported to the surface of the cell, and this complex is then capable of being recognized by T cells.
Dendritic cells (DCs) are a diverse population of morphologically similar cell types found in a variety of lymphoid and non-lymphoid tissue. Most dendritic cells are potent antigen-presenting cells specialized in initiating primary T cell immune responses. Dendritic cells generally have long, tentacle-like projections which express high levels of class II MHC molecules. Dendritic cells represent less than 0.1% of white blood cells (these are referred to as xe2x80x9cblood dendritic cellsxe2x80x9d). Dendritic cells are also found in lymphoid organs (such as the tonsil) and are referred to as xe2x80x9cinterdigitating dendritic cellsxe2x80x9d when associated with T cell areas (e.g., lymphoid organs), and xe2x80x9cfollicular dendritic cellsxe2x80x9d when associated with B cell areas. They are also known as veiled cells present in afferent lymph, as Langerhans cells in the epidermis and as dermal DCs in the dermis of the skin. One function for dendritic cells is capturing antigens and stimulating T cell response. In essence, dendritic cells xe2x80x9cpick-upxe2x80x9d antigens and migrate the captured antigens to T cells. Because dendritic cells can induce a T cell response to an antigen without other adjuvants, DCs are often referred to as xe2x80x9cnature""s adjuvant.xe2x80x9d As xe2x80x9cnature""s adjuvant,xe2x80x9d dendritic cells have attracted attention for therapeutic purposes.
PCT Publication WO 94/02156 purportedly describes a method for isolating human DCs to present antigens to induce antigen specific T cell mediated responses. Uses mentioned include cellular immunotherapy and cancer treatment. U.S. Pat. No. 5,788,963 purportedly teaches methods and compositions for use of human dendritic cells to activate T cells for immunotherapeutic response against primary and metastatic prostrate cancer cells.
An embodiment of the present invention relates to kits, e.g., diagnostic assay kits, utilizing the antibody to tumor-associated antigens and carrying out the method disclosed here.
An embodiment of the present invention may be described as a hybridoma formed by fusion of a tumor cell and a non-tumor cell, more specifically, lymphocytes and tumor cells. The hybridoma provides continuously replicating (hybrid) cells which exhibit some or all the characteristics of the non-tumor cell. The hybridomas of the present invention are used to produce or secrete antibodies which interact with cells having a dendritic morphology. Preferably, the monoclonal antibodies of the present invention produced by the hybridomas recognize cells having both a dendritic morphology and a B cell phenotype. Even more preferably the hybridoma is specific for cells which are defined herein as B-DC cells. A hybridoma in accordance with the present invention has been deposited with the American Type Culture Collection (A.T.C.C.) and has an A.T.C.C. Accession Number HB-12430 (reported with the A.T.C.C. 10801 University Boulevard, Manassas, Va. 20110-2209; U.S.A., U.S.A., Nov. 14, 1997, determined viable by the A.T.C.C. on Nov. 18, 1997).
Another embodiment of the present invention is a hybridoma comprised of lymphocytes and tumor cells wherein at least a portion of the lymphocytes and tumor cells are fused to form a hybridoma capable of producing antibodies. The antibodies preferably recognize a 220 kDa protein on the surface of cells. The 220 kDa protein located on the cells is recognized by the antibodies produced by the hybridoma and is further capable of being reduced to four subunits which consist of about 55 kDa, about 65 kDa, about 80 kDa, and about 85 kDa subunits. It is preferable that the 220 kDa protein be an antigen, and that the hybridoma be designated hybridoma 5G9 with A.T.C.C. Accession Number HB-12430. The monoclonal antibody expressed by hybridoma 5G9 (mAb 5G9) is a preferred embodiment capable of reacting with cells expressing the 220 kDa protein. It is to be understood that other monoclonal antibodies having substantially the same function (i.e., able to recognize 220 kDa proteins) are encompassed within the scope of the present invention.
Another embodiment of the present invention is a monoclonal antibody or fragment thereof which is capable of reacting with cells expressing a protein on the surface of the cell which is indicative of the cells"" morphology, preferably a dendritic like morphology. As discussed above, it is preferable that the antibodies are produced by a hybridoma cell line, most preferably hybridoma cell line 5G9, having A.T.C.C. Accession N HB-12430, or subclones thereof. It is preferable that the monoclonal antibody of this embodiment react with specific lymphoma cells, even more preferably lymphoma cells known as chronic lymphocitic leukemia cells.
Another embodiment of the present invention is a cell which is isolated from a blood cell population wherein the cell expresses the 220 kDa protein discussed above. It is more preferable that the cell expresses the protein on the surface of the cell. It is also preferred that the cell which is isolated have a dendritic morphology and even more preferably also a B-cell phenotype. Additionally, it is preferable that the cell have a function which is similar to that of classic dendritic cells. More preferably the cells react with the monoclonal antibody 5G9 or a monoclonal antibody specific for the 220 kDa cell surface protein. It is even more preferable that the cell be isolated from peripheral blood mononuclear cells and even more preferable that the cell express a phenotype which is selected from the group CD19+, CD20+, CD40+, CD83+ (after positive selection), HLA-D- hi positive, immunoglobulin xcexc chain positive, Kappa light chain positive, lamda-light chain positive, CD3xe2x88x92, CD4xe2x88x92, CD5xe2x88x92, CD10xe2x88x92, CD13xe2x88x92, CD14xe2x88x92, CD15xe2x88x92, CD16xe2x88x92, CD33xe2x88x92, CD56xe2x88x92, CD64xe2x88x92, and any combination of one or more of the above phenotypes. Even more preferably the phenotype is selected from the group consisting of CD4xe2x88x92, CD33xe2x88x92, and both CD4xe2x88x92 and CD33xe2x88x92. As used herein, cell morphology generally refers to the processes and preferably long and thin processes, of the cells which give rise to classical morphological association with dendritic cells.
Another embodiment of the present invention is a method of identifying an antigen presenting cell which is comprised of exposing the population of cells to a monoclonal antibody, preferably monoclonal antibody mAb 5G9. In this method it is preferable that mAb 5G9 be secreted by a hybridoma designated 5G9 (A.T.C.C. Accession Number HB-12430) and that the antigen presenting cell have a phenotype selected from the group above, and even more preferable that the phenotype be selected from the group CD4xe2x88x92, CD33xe2x88x92, and both CD4xe2x88x92 and CD33xe2x88x92.
Another embodiment of the present invention is an enriched cell population which is comprised of cells expressing a predetermined protein or antigen. It is preferred that the enriched population of cells have a proportion of cells having a dendritic morphology, and that the cells having a dendritic morphology appear in a higher concentration or higher purity in the enriched population than that which is endogenous for naturally occurring cell sources to which it is being compared. It is preferable that the cells having a dendritic morphology have a B cell phenotype in that they also function similar to dendritic cells. It is even more preferable that the enriched population have a portion of cells which react with monoclonal antibody 5G9 (mAb 5G9) and more preferably that they be isolated from peripheral blood mononuclear cells. As discussed herein, it is preferable that at least a portion of an enriched population of cells exhibit long and thin processes similar to dendritic cells. With regard to the protein or antigen it is preferred that the 220 kDa protein is capable of being reduced to four subunits, specifically subunits of about 55, 65, 80and 85kDa.
Another embodiment of the present invention is an isolated antigen presenting cell having dendritic cell properties and characteristics. In this embodiment of the present invention, the antigen presenting cells preferably have a morphology and function similar to that of classic dendritic cells. It is preferred that the antigen presenting cells of this embodiment be reactive with a monoclonal antibody, preferably monoclonal antibody 5G9 (mAb 5G9). It is also preferable that the antigen presenting cell be a peripheral mononuclear cell which expresses a phenotype as described above and preferably a CD83+ (after positive selection) phenotype.
Another embodiment of the present invention is a therapeutic composition which is comprised of an antigen presenting cell wherein said antigen presenting cell has dendritic cell properties and characteristics and a substantially similar function. It is preferable that the antigen presenting cell of the therapeutic composition be reactive with monoclonal antibody 5G9 (mAb 5G9) or a similar antibody which is specific for a 220 kDa protein located on the surface of a cell. As above, it is preferable in a therapeutic composition that the antigen presenting cell express a phenotype selected from the group CD19+, CD20+, CD40+, CD83+ (after positive selection), HLA-D- hi positive, immunoglobulin xcexc chain positive, Kappa light chain positive, lamda-light chain positive, CD3xe2x88x92, CD4xe2x88x92, CD5xe2x88x92, CD10xe2x88x92, CD13xe2x88x92, CD14xe2x88x92, CD15xe2x88x92, CD16xe2x88x92, CD33xe2x88x92, CD56xe2x88x92, CD64xe2x88x92, and any combination of one or more of the above phenotypes, and even more preferably that the composition include an antigen presenting cell.
Another embodiment of the present invention is a method of treating a mammal which is comprised of administering a therapeutically effective amount of a B-DC cell to the mammal, wherein the B-DC cell has both B cell phenotype and dendritic cell morphology.
Another embodiment of the present invention is a method of isolating dendritic like cells comprising reacting a population of cells with a monoclonal antibody, preferably monoclonal antibody 5G9. In this embodiment it is preferable that the cells which are to be isolated have a morphology similar to the dendritic cell, functions similar to the dendritic cell and that the cells are reactive therewith.
Another embodiment of the present invention is a method of initiating a therapeutic response which is comprised of administering to a patient an effective amount of a cell having a B cell phenotype and a dendritic cell morphology (i.e., a B-DC cell). Yet another embodiment of the present invention is a method of isolating cells having dendritic like morphology comprised of obtaining a cell sample which contains the cells having dendritic like morphology and mixing a sample of the monoclonal antibody capable of reacting with the antigen expressed by the dendritic like cells. It is preferable that monoclonal antibody have a recognition characteristic similar to that of monoclonal antibody 5G9 in that it recognizes a cell-surface antigen preferably a self-servicing antigen comprised of a 220 kDa protein. After the samples are mixed, the cells are selected so as to separate from the sample a substantially pure population of cells having dendritic like characteristics, preferably both with a dendritic like characteristic and B cell type phenotype.
In one embodiment, the diagnostic kit would conventionally include the monoclonal antibody mAb 5G9 in one or more containers, a conjugate of a specific binding partner for the antibody, a label capable of producing a detectable signal, and instructions for its use. The kit may be conjugated to a label, as is well known to the art. Various labels include enzymes, radioisotopes, particulate labels, chromogens, fluorescers, chemoluminescers, coenzymes, free radicals, and bacteriophages. Additionally the antibody may be bound to a support. The instructions for use are suitable to enable an end user to carry out the desired test. By the term xe2x80x9cinstructions for use,xe2x80x9d it is meant a tangible expression describing the reagent concentration for at least one assay method, parameters such as the relative amount of reagent and sample to be admixed, maintenance time periods for reagent/sample admixtures, temperature, buffer conditions and the like. It is within the scope of this invention to provide manual test kits or test kits for use in automated analyzers.
Specific preferred embodiments of the present invention will become apparent from the following Figures, detailed description examples and claims.